Environmental mutagens are able to induce mutations in the genomes of prokaryotes and eukaryotes in a non-random fashion with regard to the DNA sequence. An understanding of this non-random character of mutagenesis will eventually lead to better estimates of the risks presented by environmental mutagens. To study the mutational specificity in mammalian cells at the molecular level, the adeninephosphoribosyltransferase (aprt) gene from Chinese Hamster Ovary cells was chosen as a model system. This gene has been cloned previously. Specific advantages of this system include the relatively small gene size (completely contained in a 3.8 kb BamHI restriction fragment) and the well characterized selective growth procedures for aprt-deficient cells. In addition, a hemizygous cell-line (D422) is available which has a complete deletion of one of the two aprt alleles. Using this cell line, a direct correlation can be made between the mutant phenotype and the remaining aprt allele. About 100 spontaneous and 60 X-ray-induced mutants have been selected so far. To permit rapid cloning of the mutant alleles, several new cloning vectors have been constructed and methods for repetitive mamalian gene isolation have been optimized. In a preliminary test of the procedures, 30 aprt alleles were isolated, which represent 21 independent spontaneous mutants. Several of these mutants have been analyzed by rapid DNA sequencing techniques.